The development of opioid tolerance and dependence continues to be a widespread public health concern. Opioids generate addictive effects by activating G protein coupled receptors in the striatum, which leads to adaptive changes in signaling pathways. Our long-term goal is to understand the molecular regulation of striatal G protein activity due to its importance for managing drug addiction. Recent work has revealed that G protein signaling is deactivated within the cell by a family of proteins termed Regulators of G protein Signaling (RGS). This provides the cell an intrinsic mechanism to modulate signaling during receptor stimulation by drugs of abuse, however little is known about the roles of RGS in addiction. This proposal is focused on the molecular regulation of signaling reactions by essential striatal RGS proteins during the development of opioid tolerance and dependence. Recent work has shown that RGS7 and RGS9-2 bind the same adapter proteins to form macromolecular complexes in the striatum. The abundance of these RGS subunits is sensitive to morphine exposure and mouse models have revealed the differential involvement of the RGS complex subunits in shaping behavioral responses to opioids. Collectively with our preliminary data, we hypothesize the RGS complex plays an essential role in regulating MOR signaling that gates sensitivity to the central downstream effector adenylate cyclase and the generation of the second messenger cAMP. This hypothesis will be tested in two complementary aims: 1) To understand adaptations in adenylate cyclase-cAMP axis in response to opioid receptor activation and the role of RGS complex in this process, 2) To characterize the mechanistic role of individual subunits in the RGS complex in controlling opioid signaling. Our approach will utilize optical assessment of live brain slice preparations from a multitude of genetic mouse models in addition to advanced cell-based assays. The conclusion of these studies will greatly expand our knowledge of opioid signaling plasticity, roles of RGS proteins in addiction, and evaluation of a novel mouse model.